STOOL CONCENTRATION

METHODS
Dr.P.B.PRAVEENKUMAR
FIRST YEAR POST GRADUATE
DEPARTMENT OF MICROBIOLOGY
THANJAVUR MEDICAL COLLEGE
 When will we go for concentration of the
stool specimen????
• If the parasite output is low.
• Whenever epidemiological analysis is needed.
• To assess prognosis.

NOTE: Eggs, cysts and larvae can be seen.

Trophozoites get destroyed.
 TYPES OF CONCENTRATION METHODS
• Sedimentation
• Floatation
 SEDIMENTATION METHODS
• Eggs and cysts settle down at the bottom
following centrifugation.
TYPES:
• Simple sedimentation technique
• Formalin-ether concentration technique
• Formalin-ethyl acetate concentration technique
• Formalin-acetone sedimentation technique
• Formalin-hemo-de sedimentation technique
 PRINCIPLE
• Concentration of stool specimen by
centrifugation
• Concentrated at the bottom of the tube since
they have greater density than the suspending
medium
 SIMPLE SEDIMENTATION
• Significant amount of faeces is thoroughly
mixed with 10-20 times of tap water and
shaken several times.
• Then allow that diluted faeces sample to settle
it in a conical flask for 1 to 2 hours.
• Repeat several times till the time supernatant
fluid is clear.
 PROCEDURE
(Modified Ritchief’s method by Ridley and Hagwood, 1956)

1) 1 gram of feces is emulsified in 7 ml of 10%

formol-saline and kept for 10 minutes for
fixation.
2) Then the mixture is transferred into a conical
centrifuge tube through two layered gauze.
3) The filtrate is collected in a centrifuge tube.
4) 3 ml of ether is added in centrifuge tube and
close the tube using stopper, mix vigorously
for 1 minute.
 PROCEDURE (cont.)
5) Then centrifuged at 2000 rpm for 2 minutes
and allowed to settle. (Centrifugation should
be done after stopper removal)
6) Four layers are formed.
6A(Top)Ether
6BPlug of debris
6CClear layer of formalin
6D(Bottom)Sediment
 PROCEDURE (cont.)
7) The debris is loosened using a glass rod and
cleared.
8) Supernatant fluid is decanted.
9) The deposit is poured after shaking in a glass
slide to look for saline or iodine mount.
 ADVANTAGES
• Increased sensitivity to detection of ova and
cysts since formalin fixes the ova and cysts.
• Size and shape of parasite is maintained.
• Inexpensive and easy to perform.
• Faecal odour is removed using formalin.
• Ether dissolves faecal fats.
• Even formalin kills faecal parasites, so no
laboratory acquired infection.
 DISADVANTAGES

TROPHOZOITES CANNOT BE DETECTED

Since ether is explosive, what are all other
alternatives???
• Ethyl acetate
• Acetone
• Hemo-de (Clearing agent)
 If we are going to do this sedimentation
technique for formalin preserved stool..THEN???

SKIP STEP 1
(STEP:1 - 1 gram of feces is emulsified in 7 ml of
10% formol-saline and kept for 10 minutes for
fixation)
 If the stool contains lot of mucus, then
anything to do????

SKIP STEP 2
(STEP – 2 - Then the mixture is transferred into a
conical centrifuge tube through two layered
gauze)
 If the stool is PVA preserved, then....

MODIFICATION OF STEP 1
(STEP:1 - 1 gram of feces is emulsified in 7 ml of
10% formol-saline) No need to wait for 10
minutes, immediately go for STEP 2
BAERMANN CONCENTRATION METHOD
• Used to see the fecal specimen containing
small number of strongyloides larva.
• PRINCIPLE: Strongyloides larva migrates from
cooler to warmer area.
 PROCEDURE
• 5 Grams of feces is placed on the gauze.
• Funnel is filled with warm water and left 1-2
hours to give time for strongyloides larva to
come out from feces.
• After 1-2 hours, clamp of funnel is opened, 7-8
ml of water is collected in beaker.
• Larva migrate downards bottom of the fluid.
• After centrifugation, sediment should be
examined microscopically.
MODIFICATION OF BAERMANN TECHNIQUE

• Instead of funnel here we are using test tube with

rubber stopper.
• Perforation of rubber stopper to allow insertion of
plastic pipette tip to take out supernatant fluid.
• The tube containing faecal suspension is inverted
over another tube containing 6 ml of saline and
incubated at 37 degrees celsius for 2 hours.
• After centrifuge, saline solution is screened for
larvae.
 FLOATATION TECHNIQUE
• Faecal material dissolved in solution of higher
density than that of eggs.
• In this method, eggs will float in superficial
part of the fluid.
• All eggs will float in solution except
(i) Unfertilized eggs of Ascaris lumbricoides
(ii) Eggs of Taenia saginata and Taenia solium
(iii) Eggs of all intestinal flukes.
TYPES OF FLOATATION TECHNIQUE
• Simple / Saturated salt floatation technique
• Direct Centrifugation Floatation / D.C.F
technique
• Zinc sulphate centrifugal floatation technique
 SIMPLE SATURATED SALT FLOATATION
TECHNIQUE
Materials required:
• Glass (or) metal container of 15-20 ml capacity
• Glass slide of size 3*2 >> 3*1
• Saturated salt solution of specific gravity of
1.200
• A sheet of glass on which the glass container is
placed.
 PROCEDURE
• 1 ml of faeces is taken in container along with
few drops of salt solution.
• Mix it with glass rod or stick to prevent uneven
emulsion.
• Now, add 15 to 20 ml salt solution along with
mixing using glass rod.
• Any coarse matter floats up means, remove it
without any fear (EGGS WILL TAKE MORE TIME
TO FLOAT)
 PROCEDURE (cont.)
• At this stage, container is placed at clean glass
sheet (EVENFUL surface)
• The final filling of container should be done by
dropper till the time a convex meniscus is
formed.
• A glass slide is carefully laid on the top of the
container so that centre of the slide is in
contact with the fluid.
 PROCEDURE (cont.)
• The preparation is allowed to stand for 20 – 30
minutes.
• After which, glass slide is quickly lifted, turned
over smoothly so as to avoid spillage.
• Examined under microscope after putting
coverslip.
 LANE’S DIRECT CENTRIFUGAL FLOATATION
TECHNIQUE (D.C.F)
Materials required:
• Special bucket (For containing the centrifugal
tube) with flat bottom and special guard to
keep cover slip in position.
• Special cover slip having 19mm square by 0.5
mm thickness.
• Lane’s centrifugal machine.
 PROCEDURE
• 1 (or) 2 gram of faeces mixed with water in a Clayton
Lane’s centrifugal tube.
• Centrifuge the mixture for 2 minutes at 1000 rpm.
• Then supernatant fluid is poured off.
• Add saturated salt solution with specific gravity of
1.200 till the level of brim and then thick cover slip is
put on.
• Centrifuge at 1000 rpm for 2 minutes again.
• Cover slip is quickly lifted off and examine that one
drop undersurface by means of hanging drop method.
 FAUST ZINC SULPHATE CENTRIFUGAL
FLOATATION TECHNIQUE
1) 1 gram of faeces is emulsified in 10 ml of
distilled water and kept for 10 minutes for
fixation.
2) Then the mixture is transferred into a conical
centrifuge tube through two layered gauze.
3) The filtrate is collected in a centrifuge tube.
4) Centrifuge the tube at 2500 rpm for 1 minute
repeatedly till the time that supernatant fluid
is clear.
 PROCEDURE (cont.)
5) Add 3-4 ml of a 33% zinc sulphate solution having
specific gravity 1.800 along with stirring till top
of the tube. (For formalin preserved stool, zinc
sulphate of specific gravity 1.200 is used)
6) Again centrifuge at 2500 rpm for 1 minute.
7) The surface film is removed by platinum loop of
5 mm, place it on clean glass slide and then
focus it in microscope after placing cover slip.
The sediment also mounted and examined.
ZINC FLOATATION TECHNIQUE
 SHEATHER’S SUGAR FLOATATION
TECHNIQUE
• Used for detection of Cryptosporidia infection.
THANK YOU